Zhao Hongqing, Sun Jiachen, Wu Yating, Zhang Junbo, Shen Chuan'an
Nanbu County People's Hospital, Nanchong City, Sichuan Province, 637300, China.
Jinzhou Medical University, No.82 Songpo Road, Guta District, Jinzhou 121001, Liaoning Province, China.
Regen Ther. 2023 Jul 20;24:245-255. doi: 10.1016/j.reth.2023.07.003. eCollection 2023 Dec.
The physiological process of wound healing is dynamic, continuous, and intricate. Nowadays, full-thickness burn wounds are treated by autologous skin transplantation. Unfortunately, when substantial burns develop, there are fewer donor sites accessible, making it difficult to satisfy the requirement for large-scale skin transplants and increasing the risk of patient mortality. This study investigated the possibility of using a newly created hypoimmunogenic epidermal cell sheet to heal skin wounds.
Transfection with lentivirus was used to generate Keratinocytes (KCs) that overexpress Indoleamine 2,3-Dioxygenase (IDO). Western blotting and quantitative polymerase chain reaction were used to measure IDO levels. To evaluate the function of IDO keratinocytes, CCK-8 and Transwell assays were performed. In cell sheet induction media, KCs and Fibroblasts (FBs) were cultured to yield epidermal cell sheets. The full-thickness skin excisions of BALB/c mice were transplanted with epidermal cell sheets. To assess the tumorigenicity of IDO keratinocytes, BALB/c nude mouse xenograft models were also used. CD3 and CD31 immunofluorescence labeling of wound tissue on day 12 to identify T lymphocyte infiltration and capillary development. ELISA measurement of IL-1 and TNF-α concentrations.
IDO keratinocytes dramatically enhanced the expression levels of IDO mRNA and protein, as well as the amount of kynurenine in the conditioned media of IDO keratinocytes, compared to the Control and NC groups. CD8 T cell apoptosis was considerably greater in the IDO group than in the Control and NC groups. Nevertheless, the proliferation and migratory capabilities of IDO keratinocytes were not substantially different from those of the Control and NC groups. In vitro cultivation of the hypoimmunogenic epidermal cell sheet was effective. In vivo transplantation experiments demonstrated that IDO epidermal cell sheets can effectively promote wound healing without tumorigenicity, and IDO epidermal cell sheets may promote wound healing by decreasing the expression levels of inflammatory factors (TNF and IL-1) in wound tissue, decreasing CD3 T lymphocytes, and increasing infiltration and new capillaries in wound tissue.
In this study, we successfully constructed the hypoimmunogenic epidermal cell sheet and demonstrated that the hypoimmunogenic epidermal cell sheet could accelerate wound healing.
伤口愈合的生理过程是动态、连续且复杂的。如今，全层烧伤创面通过自体皮肤移植进行治疗。不幸的是，当出现大面积烧伤时，可用的供皮区较少，难以满足大规模皮肤移植的需求，且增加了患者死亡风险。本研究探讨了使用新创建的低免疫原性表皮细胞片来愈合皮肤伤口的可能性。
采用慢病毒转染生成过表达吲哚胺2,3-双加氧酶（IDO）的角质形成细胞（KCs）。使用蛋白质免疫印迹法和定量聚合酶链反应来测量IDO水平。为评估IDO角质形成细胞的功能，进行了CCK-8和Transwell实验。在细胞片诱导培养基中培养KCs和成纤维细胞（FBs）以生成表皮细胞片。将BALB/c小鼠的全层皮肤切除创面移植表皮细胞片。为评估IDO角质形成细胞的致瘤性，还使用了BALB/c裸鼠异种移植模型。在第12天对伤口组织进行CD3和CD31免疫荧光标记以识别T淋巴细胞浸润和毛细血管形成。通过酶联免疫吸附测定法测量IL-1和TNF-α浓度。
与对照组和阴性对照组相比，IDO角质形成细胞显著提高了IDO mRNA和蛋白的表达水平，以及IDO角质形成细胞条件培养基中犬尿氨酸的含量。IDO组中CD8 T细胞凋亡明显高于对照组和阴性对照组。然而，IDO角质形成细胞的增殖和迁移能力与对照组和阴性对照组相比无显著差异。低免疫原性表皮细胞片的体外培养是有效的。体内移植实验表明，IDO表皮细胞片可有效促进伤口愈合且无致瘤性，IDO表皮细胞片可能通过降低伤口组织中炎症因子（TNF和IL-1）的表达水平、减少CD3 T淋巴细胞以及增加伤口组织中的浸润和新生毛细血管来促进伤口愈合。
在本研究中，我们成功构建了低免疫原性表皮细胞片，并证明低免疫原性表皮细胞片可加速伤口愈合。
